Influence of the interval between removal and freezing of muscle samples on muscle fibre size with special reference to sample size and fibre type

To study the influences of the interval between removal and freezing of a muscle sample on fibre size, rat hindlimb muscles (soleus and extensor digitorum longus, EDL) were cut into three or five pieces and frozen at different intervals, cross-sectioned, stained and analysed morphometrically. As in a previous study (Larsson & Skogsberg 1988), a pronounced interval-dependent change in soleus muscle fibre size was observed, the fibres in soleus muscle samples frozen immediately (within 15 s) being significantly (P less than 0.01-0.001) larger than those samples frozen 4 or 10 min after removal. However, the interval-dependent change was only noted in small muscle samples, i.e. those where the maximum muscle fibre length was 6.5 mm, while samples in which the fibres were approximately twice this length showed no change. In the slow-twitch soleus, type I fibres predominate, and type II fibres were often too few in one or more cross-sections in each animal to allow reliable measurements of fibre size. The fast-twitch EDL was therefore studied, and the results indicated an interval-dependent change in fibre size which was differentiated according to enzyme-histochemical type, i.e. there was a significant change in type I and IIA fibres but not in type IIB. It is concluded that the interval between removal and freezing of small muscle samples, as in percutaneous muscle biopsies in man, is of vital importance for fibre size, and that this interval should be at least 2-4 min.